Loss of function mutations in PINK1, a mitochondrial kinase, are associated with recessive parkinsonism. Previous work has shown that increased expression of wild type, but not mutant, PINK1 can protect cells against several mitochondrial toxins. The kinase activity of PINK1 is critical for this function, suggesting that the protective aspects are mediated by as an yet undefined signal transduction pathway. Work in Drosophila models, in particular, has defined a pathway that includes both PINK1 and parkin, another gene for recessive parkinsonism in humans, that is important for maintaining the morphological integrity of mitochondria. In a recent study, we have characterized the effects of loss of function of PINK1 in human cells in culture. We found that PINK1 deficient human cells, compared to controls, had a number of linked mitochondrial phenotypes and, using a number of live cell imaging approaches, we were able to determine which effects were primary and which were consequential of each other. A relatively early in this system appears to be a loss of mitochondrial membrane potential, which is normally maintained by the actions of the respiratory chain complexes in the inner mitochondrial membrane but can be disrupted by a number of stressful stimuli. We were able to show that subsequent to depolarization, the calcium dependent phosphatase calcineurin is activated. This tends to dephosphorylate a key play in mitochondrial shape and dynamics, dynamin-related protein 1. Any morphological changes in mitochondria are therefore downstream of biochemical events. We are currently attempting to understand why mitochondria in PINK1 deficient cells have depolarized mitochondria as if this is a primary event it may represent an important clue for PINK1 function. We are also expanding this work to examine other genes involved in recessive parkinsonism. As well as understanding the cellular phenotypes related to PINK1, we have also begun to characterize how these might relate to the biochemistry of the protein. PINK1 binds to two interacting partners, an atypical calcium-dependent GTPase, Miro-2, and the adapter protein, Milton-1, likely at the cytoplasmic face of the mitochondria. We were able to show that Miro/Milton when overexpressed can rescue the partially fragmented phenotype of PINK1 deficient cells, although mitochondria tended to aggregate under these circumstances. This data shows that using quantifiable cellular phenotypes is a useful approach for validating and exploring protein-protein interactions for PINK1, which we plan to expand in future experiments.